Essentially all developing and adult tissues contain one or more populations of stem cells and/or progenitor cells (progenitors) that play a role in the continued maintenance of health of the tissue through remodeling activity. Such stem cell and progenitor populations also contribute to new tissue formation in the event of injury, and represent an essential resource in tissue engineering strategies seeking to repair, augment, replace or regenerate tissues that may be lost due to injury, disease, or degenerative or aging processes.
For example, bone repair requires osteogenic connective tissue progenitors (CTP-Os). In settings where the local population CTP-Os is sufficient, they may be effectively “targeted” using scaffolds or factors, such as bone morphogenic proteins to summon the CTP-Os to where they are needed. However, in settings where the levels of local CTP-Os are suboptimal, optimizing the bone healing response requires transplantation of CTP-Os from an alternative source. Many preclinical studies demonstrate improved graft performance when CTP-Os are added, even to small graft sites in young healthy animals, supporting the premise that the CTP-O population is suboptimal in virtually all clinical settings and that optimal performance from any osteoconductive or osteoinductive material may require augmentation with local CTP-Os.
As a result of the potential importance of progenitor cell populations in maintaining or defining the current health of tissue, and as a resource for cell therapy strategies, methods for the harvest, isolation, assay, characterization, processing and transplantation of progenitor cells have exceptional value, and are expected to be the focus of many advances in health care diagnosis and treatment modalities.
Data available to date from many organ systems has demonstrated that the concentration and prevalence of progenitor cells is generally very low, and varies widely from tissue to tissue and individual to individual. Investigators have speculated that the concentration and prevalence of progenitors are a reflection of the current state of health of a tissue and may also predict the future health of a tissue or individual. As a result, they are likely to have important implications in diagnosis and prediction of disease as well as in the treatment of disease.
Adult stem cells present in native tissues tend to be distinctly different from the much more numerous population of mature cells in native tissue with respect to both morphological as well as chemical and biological properties. Each of these has been used in reported methods for progenitor cell isolation. Cell size, cell density, and granularity have been used as means of enrichment using density separation and countercurrent elutriation. Membrane bound surface markers in the form of membrane bound protein antigens that are uniquely presented on selected stem cell and progenitor populations can be targeted using antibodies. For example, the presence or absence of CD34, c-kit, Sca1 and other markers, alone or in combination, have been used to isolate and fractionate hematopoietic stem cells from marrow and other tissues using fluorescent activated cell sorting (FACS), magnetic separation or affinity columns. adult stem cells also tend to express novel markers and patterns of gene expression. Underlying gene expression, while generally silent, can and has been converted through viral transfection vectors into fluorescent reporters that can be used as a basis for isolation. Finally, cell function, such as the presence of a selective ABC membrane pumps have been identified as a unique feature of several stem cell populations, and have been used to isolate what has been referred to as “side population” cells or SP cells, from marrow and other tissues.
Many markers have been proposed for positive selection of human osteogenic connective tissue progenitors, such as STRO-1, STRO-1 with VCAM-1, and CD antigens 9, 10, 13, 18, 29, 44, 49a, 54, 90, 105, 146 and 166. See Simmons et al., Blood (1991) 78(1), p. 55-62. Alkaline phosphatase and osteocalcin are also markers of some circulating CTP-Os. However, most of these markers are also present on other cell populations, limiting their usefulness for positive selection of CTP-Os. While positive markers have been elusive, CTP-Os may also be differentiated from the vast majority of marrow cells based on markers that they do not express. For example, CTP-Os are negative/dim for CD45 and many other hematopoietic markers. Hematopoietic markers therefore provide possible tools for CTP-O enrichment by negative selection or depletion of non-osteogenic cells.
The most common method of isolation of stem cell and progenitor populations exploits the biological capacity of these cells to proliferate, and particularly the capacity of adult stem cells to proliferate under some conditions in a manner that exponentially increases their number while at the same time preserving one or more desirable biological capacities (e.g. the ability to repopulate bone marrow in an animal that has been depleted of hematopoietic stem cells, or the ability to form new bone tissue in vivo). This strategy of in vitro expansion and purification has been used to prepare populations of cells defined variably as bone marrow stromal cells (MSCs), mesenchymal stem cells (also “MSCs”), mesenchymal progenitor cells (MPCs), multipotent adult progenitor cells (MAPCs), tissue regenerating cells (TRCs), muscle-derived progenitor cells (MDPCs), adipose-derived stem cells (ADSC), and others.
It has long been recognized that while culture-expanded stem cell populations can be prepared that retain desirable biological capabilities, these populations differ significantly from the population of adult stem cells that are present in native tissue from which they are derived. Differences may be expressed in cell size, cell cycle state, expression of markers, and gene expression, as well as intrinsic biological behavior such as responses to growth factors. Furthermore, the use of culture-expanded cells is associated with the need for delay between the harvest of founding cell population and the ultimate use of the resulting expanded cell population. This delay adds significantly to the cost and also to the inconvenience of using culture-expanded cells, because the patient must be exposed to separate procedures; first to collect founding cells, and second to implant cells after in vitro expansion. In addition, in vitro expansion adds the potentially significant risks of bacterial or viral contamination of cells while in vitro, in vitro selection of cells with undetected undesirable biological properties (e.g. tumor forming cells) and even contamination with other cells or mislabeling with respect to the donor of origin.
The rapid isolation and processing of adult stem cells isolated from tissues of an individual at the time of a single therapeutic procedure has great potential value, and avoids many of the drawbacks of culture expanded cell populations cited above. However, rapid processing of freshly isolated cells has itself a number of drawbacks. First, adult stem cells are typically very few in number. The prevalence of progenitor cells (tissue forming cells) within a given tissue can be as high as one in 100 cells, but also as low as one in 1,000,000 cells (or less). Second, stem cells and progenitor populations in native tissues are generally very heterogeneous, in contrast to the relatively homogenous culture-expanded stem cell and progenitor cell populations. No one feature or combination of features can define all adult stem cells in a given tissue. In fact, one must expect that a given tissue will provide a diverse population of adult stem cells that represent cells from multiple stem cell niches within the tissue, each representing a different compartment or niche for the tissue forming cell populations within that tissue. See Muschler et al., J. Biomed. Biotechnol. (2003); 2003(3), p. 170-193.
Rapid processing has two important advantages, however. First, processing strategies can be designed to take advantage of characteristics of freshly isolated cells that may not be preserved when cells are expanded in vitro. Second, due to the high potential that adult stem cells have for proliferation, transplantation of a relatively small number of adult stem cells into a wound in an environment in which cells are likely to survive can result in important and clinically significant improvement in biological outcome. In fact, removal of competing and non-tissue forming cells may be just as important, if not more important, to the performance of transplanted progenitor cells as transplanting them in large numbers. For example, several recent reports have shown that as little as a 3-4 fold increase in the concentration of osteogenic connective tissue progenitor cells can result in significant improvement in bone formation and in union rate in settings of spinal fusion and in settings of bone grafting in long bone defects. See U.S. Pat. Nos. 6,049,026 and 6,723,131, issued to Muschler. Removal of competing cells may eliminate a source of growth factor or signaling molecules that are maladaptive to proliferation and new tissue formation, such as inflammatory cytokines that may stimulate apoptosis (cell death). Removal of competing non-tissue forming cells may also dramatically improve the likelihood that transplanted progenitor cells will survive following transplantation, by reducing local consumption of oxygen and other nutrients. See Muschler et al., J. Bone Joint Surg. Am. (2004) July; 86-A(7), p. 1541-58.
Bone and marrow tissue, including bone marrow harvested using the minimally invasive method of aspiration contains a heterogeneous population of cells, including adult stem cells capable of regenerating connective tissues, blood cells, blood vessels, bone, cartilage, fat, marrow stroma, muscle, tendons, ligaments and other fibrous tissue. These populations include multipotent cells which are individually capable of giving rise to progeny along all three germ lines (i.e. ectoderm, endoderm and mesoderm), pleuripotent progenitors capable of giving rise to progeny that may contribute to multiple mature cell types (e.g. bone, cartilage, fat), and mono- or uni-potent progenitors that are committed to progeny of only one lineage. These diverse and versatile cell sets are used extensively in research settings as well as clinically in bone grafting and tissue engineering endeavors. Bone marrow aspirations offer many advantages as a cell source. In particular, they result in very low morbidity to the patient and provide cells in single cell suspension that can be manipulated and processed using only an anticoagulant, without the need for enzymatic digestion that may modify the cell surface.
One method to increase the concentration of bone forming progenitors is density separation, which is available through use or modification of devices designed for clinical preparation of platelet rich plasma. See Hernigou et al., J. Bone Joint Surg. Am. (2005) July; 87(7), p. 1430-1437. Density separation can increase the CTP concentration 4-8 fold, but is relatively non-selective, and does not change osteogenic CTP prevalence.
Focusing on bone forming progenitors in bone marrow, and using the “gold standard” method for assay of progenitor populations (i.e., the colony forming unit (CFU) assay), investigators have found wide variation between individuals and between individual aspirate samples, but a mean prevalence of osteogenic CTPs (CTP-Os) of approximately one in every 20,000 cells. Utilizing this CFU assay, the unique property of many CTP populations (e.g. CTP-Os) to preferentially and rapidly adhere to selected surfaces has been investigated, particularly with regard to surfaces that can be created or utilized in a porous implantable matrix or scaffold. This investigation resulted in the recognition of the process of selective retention, which has been used to develop the Cellect™ graft preparation device, now manufactured and marketed by DePuy Spine Inc.
Selective Retention (SR) involves passing a cell suspension through a porous matrix and uses the intrinsic attachment behavior to retain CTP-Os in the matrix, while non-adherent cells pass through the matrix in the effluent solution. SR has been used to enrich CTP-Os as much as 16 fold. See Muschler et al., Clin. Orthop. Rel. Res. (2003) 407, p. 102-118. Current scaffolds used for SR (e.g., bone matrix, TCP ceramic) will retain 80-90% of CTP-Os and only 20-30% of other nucleated cells, resulting in a 3-4 fold increase in the CTP concentration and also a 2-3 fold increase in CTP prevalence by removing 70-80% of potentially competing cells. Thus, while effective, the principal limitation of selective retention is the fact that many of the vastly more abundant non-progenitor cells also bind to the same surfaces, and although they are less adherent, they occupy a much larger fraction of available surface. SR processing has the advantage of requiring relatively simple instrumentation and a minimum of reagents, but is far from being optimized. Even in retained “CTP enriched” populations, CTP-Os represent only a small fraction of the retained cells (mean±0.05%).
Accordingly, there remains a need for a more selective marker and/or additional separation techniques that can be used to purify or enrich adult stem cells from the animal tissues in which they are found.